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To determine whether salamanders f je.tteu with hcmioholinium for .`celts were permanently poisoned, we amputated the regenerating leg and al * ed it to regrow without further . .".-'..^ration of a drug. Regeneration . ..n identical to that in controls, indi- ...hng that t!ie clTect of hemicholinium ,:s reversible. To demonstrate that the ir. he.l amputation did not stimulate sut'seeuent regeneration we measured the growth rates 10 weeks after the first amputation and similarly after a r,.amputation. The first and second rates of regeneration were identical in each of two salamanders studied. We then considered that treatment with hemicholinium might inhibit growth be cause 25 percent of the body weight was lost during treatment. For that study, the hind limb of an untreated salamander was amputated, but no food was given to the animal during the 10-week regeneration period. Despite a weight loss of 50 percent, the rate of limb growth was not different from that in previous controls. " The diminished growth rate in treated salamanders correlated well with the microscopic findings. Salamanders treated for 5 weeks with hemicholinium showed only about 30 mitotic figures in the median sagittal section of the regenerate. However, more than 400 were seen in similar sections from con trols. Colchicine (2 mg/kg) was in jected into both groups 12 hours before the salamanders were killed so that mitotic figures would accumulate and could be counted. Evidently hemicho linium did not inhibit regeneration by means of an effect like that of colchi cine, fc r far fewer mitotic figures were seer, among treated specimens than among controls. Furthermore, the fact that the mesenchymal cells were not en larged argues against the possibility that hemicholinium blocks cell division at an earlier stage. The drug also prevented the massive dedifferentiation of muscle tissue normally seen in sections just proximal to the amputation site. In un treated amputees the neurilemmal nuclei increased in number and en larged with dedifferentiation of the nerve invading more proximal parts, but treated animals showed virtually no nerve changes. Gross and microscopic examinations showed that the vascu larity of these regenerating stumps was also strikingly diminished. ij: Since the salamanders were main tained in a partially paralyzed or paretic state by treatment with hemicholinium, it is possible that disuse of the stump could account for the observed inhibi- PC 6- L,'+. tion of growth. However, in one experi than normal but which then return to- ment in which triethylcholine (400 mg/ '*""Nrd normal during the period of early kg) was administered, the rate of re -uTercntiation. generation was nearly 72 percent of As yet we have no way of deciding control, despite the fact that this drug whether treatment with hcmicholir,:.i,i- paralyzed the animal more effectively retards growth primarily by blocking than hemicholinium did. Botulinum synthesis of acetylcholine or by func toxin, which also produces paralysis, tioning like curare (9). Wc have at has been reported (6) not to delay re tempted to rule out the latter effect by generation of the salamander limb. Fur simultaneously administering choline thermore several changes were observed (10:1 molar ratio) and hemicholinium. which cannot relate to the paralytic However, the combination proved toxic. effect of drug treatment After 5 weeks A direct inhibition of mitosis by hemi of treatment with hemicholinium the cholinium has not been excluded, but taste buds of the salamander showed in view of the ability of drug treatment regressive and degenerative changes. to mimic in the salamander some After 19 weeks of treatment the taste features of dysautonomia, a disease buds disappeared, but they returned characterized by a cholinergic defect, when treatment was discontinued. Chil hemicholinium appears to act as a cho- dren with dysautonomia not only show linolytic agent. a parasympathetic insufficiency with a F. Kur growth disturbance but also a lack of A. Smith lingual taste buds (7). Departments of Pharmacology and There is much evidence supporting Anesthesiology, New York Medical the hypothesis that acetylcholine is a College, New York 10029 neurotrophic factor (5), but there are also compelling reasons against its ready acceptance. Singer (5) reports that microinfusion of atropine into the regenerate halts limb growth. On the other hand, infusion of acetylcholine into a denervated limb does not restore its regenerative capacity. However, an infusion cannot duplicate neural activ ity. A more cogent reason for excluding acetylcholine as the growth mediator is that the concentration of the sub stance in sensory nerves is negligible compared to that in motor nerves. Yet the sensory nerve is far more capable of influencing regeneration than is the motor component. On the other hand, regenerating tissue contains concentra References and Notes 1. M. Singer, Quart. Rev. Biol. 27, 169 (1952). 2. E. G. Butler and O. E. Schotte, J. Exp. Zool. 88, 307 (1949). 3. D. B. Drachman, Symposium on Growth of the Nervous System, G. E. W. Wolstenhome and M. O'Connor, Eds. (Churchill, London. 1968), pp. 251-273; D. B. Drachman, Science 145, 719 (1964). 4. J. Dancis and A, A. Smith, N. Eng. J. Med. 274, 207 (1965). 5. C. Riley, Advart. Pediat. 9, 157 (i*>57). 6. D. Drachman and M. Singer, died in Neuro science Research Progress 3:<Ue'.ln 7, L. Guth, Ed. (Brookline, Mas.'.uchjsei.is, i963), p. 35. 7. A. Smith, A. Farbman, J. Science 147, 1040 (1965). 8. M. Singer, in Developing Cell Systems n Their Control, D, Rudnick, Ed. (Ronaid, New York, 1959), p. 115-133. . 9. R. E. Thies and V. B. Brooks, Fed. Proc. 20, 569 (1961). 10. FJ1. is a fellow of the Dysautonomia As* sociation of New York. tions of acetylcholine which are greater 20 July 1970; revised 1 October 1970* a Polychlorinated Biphenyl: Interaction with Duck Hepatitis Virus Abstract. Ten-day-old mallard ducklings fed a polychlorinated biphenyl at concentrations of 25, 50, and 100 parts per million for 10 days suffered no apparent clinical intoxication. Five days later these birds were challenged with duck hepatitis virus, and they suffered significantly higher mortality than birds which were not exposed to the polychlorinated biphenyl. Polychlorinated biphenyls (PCB) along with DDE [l,l-dichloro-2,2-bis(p-chlorophenyl)ethane] are reported to be the most abundant of the chlorinated hydrocarbon polllitants in the global ecosystem (f). Despite the fact that PCB's have been in wide use since 1930 (2), they remained eco logically inconspicuou until 1966 when a Swedish chemist reported their presence in the tissues of pike and caber wildlife (5). Since then, their presence has been reported in additional wildlife of Europe and North America (/, 4). These compounds are interesting because they are an important source of interference in the chemical detec tion of DDT and its metabolites (5) and 1314 DSW 255267 SCIENCE, VOL. 170 STLCOPCB4060958 :vv.i.su- ihi". are > poteuliitl hazard in cnviruniuont; imi a weight basis ' 'H have been shown to . ,c .'...,!-:.-i-Jeg.-;n!ing potential about ;jve iimos ih.at of /;,/?'-DDE or techni cal grtulc DDT [l,l,l-trichloro-2,2-`bis(/j-chlorophenyl) ethane] (/). As part of an investigation of possi ble interactions between organochlorine pollutants and infectious diseases, this study was initiated to determine whether any interaction, antagonistic or synergistic, occurs with PCB and duck hepatitis virus (DHV) in mallards (Anas platyrhynchos). Four separate diets, three with PCB and one without, of a standard duck starter ration were fed to groups of 10-day-old mallard ducklings. The three PCB diets contained Aroclor 1254 (in a corn oil premix) concentrations cal culated to be 25, 50, and 100 parts per million (ppm). These diets were fed to live groups of 25 ducklings each, three principal and two control groups (Table 1). All birds were leg-banded with colorcoded, numbered bands, weighed, and fed their respective diets for 10 days. On day 11 each bird was weighed again, and every fifth bird from each treatment group was killed. Tissues were collected for residue analysis, his tology, and liver weights. During the 10-day feeding trial, no birds died or became clinically ill, although duck lings in the group given 100 ppm PCB were noticeably hyperexcitable after day 3 as were those in the 50-ppm group 4 days after treatment. Body weights of birds receiving PCB in their diet were significantly heavier at the end of the 10-day feeding trial than those fed the diet without PCB (Table 1). The heaviest body weights occurred in birds fed the highest con centrations of PCB, and the trend ap peared to be linear. Differences in body weights among treatment means were highly significant (F = 20.30, d.f. = 4, 119) when tested by analysis of covariance. No statistical comparisons between individual treatment means were made. A similar analysis of liver weights (determined as a percentage of final body weight) disclosed highly sig nificant differences among treatment means (F = 5.82, d.f. = 4, 18). These differences appeared to be within groups treated with PCB rather than between treated and untreated groups (Table 1). The small sample sizes (N = 5) necessitates caution in interpretation of liver weight data. No interpretation of differences among body and liver weights are offered at this time. I able I. I inul mo.m bmlv tit I liver wi ii'.inv of mallard ducklings after being feil I'CIJ (Aroclor 1254, Monsanto, St. Louis) for 10 jays. Numbers in parentheses are the number of birds used. The liver and body weights were adjusted by covariance for a common starling mean; the liver weight was determined as percentage of body weight. The final con trol did not receive cither PCB or DHV. Treatment PCB (ppm) Body weight (g) Liver weight (g) PCB 4- DHV PCB 4- DHV PCB -f DHV DHV control Control 100 50 25 0 0 381.6 (25) 368.8 (25) 352.8 (25) 315.2 (25) 309.6 (25) 0.046 ( 5) .037 (5) .029 (5) .036 (4) .034 (5) Twelve days after the experiment be gan, birds were placed in crates and transported approximately 5 miles to isolation units where they were main tained on PCB-free feed and water for 3 days before being challenged with DHV. No birds died or became clinically ill during the interim period between PCB feeding and virus chal lenge although two birds were lost from the 100-ppm group due to accidents. On day 15 of the experiment all but the untreated controls (birds not re ceiving either PCB or DHV) were in oculated intraperitoneally with 1.5 1 I),,, (.Ml piTc.v.i , lb i,-.. i or ducks) of DilV j'vr bird, in- .rst birds died about 47 hour.-, after inoculation. Mortality was recorded hourly for the remainder of the 80- hour experimental period with one ex ception; there were no observations be tween hours 63 through 69. The onset of mortality in all groups given PCB plus DHV began at least 8 to 16 hours before that in the control group treated with the virus only (Fig. 1). In addi tion, mortality levels occurring among PCB-plus-virus treatment groups at the end of the experiment were significantly higher at the .01 level of probability than for ducklings receiving the virus only (chi square = 7.49, d.f. = 1). There was no significant difference I,-, mortality among ducklings receiving r.e different concentrations of PC If pies virus (chi square = 3.79, d.f. = 2). No birds in the untreated group died (Table 2). All birds that died during the experi ment had gross pathognomonic i:\vr lesions, indicating typical duck nvp..- titis infection (6). No other gross lesions, with the exception of varying amounts of edema within the peri cardium and thoracic cavity of some Table 2. Mortality among mallard ducklings caused by DHV during an 80-hour eJt'perimenia; period. No observations were made between 31 to 47 and 62 to 70 hours. The final control did not receive either PCB or DHV. Treatment PCB (ppm) No. of deaths/ total sample . Mortality Percentage Onset (hours) Cessation (hours) PCB 4- DHV 100 8/18 44.4 31--47 75 PCB 4- DHV 50 13/20 65.0 55 76 PCB 4- DHV 25 7/20 35.0 31-47 75 DHV control 3/21 14.3 62-70 73 Control 0/20 0.0 Fig. 1. Mortality among mallard ducklings exposed to both PCB and DHV. is only, or neither agent, (a) 100 ppm PCB 4- DHV; (b) 50 ppm PCB -f DHV: i. ppm PCB 4- DHV; (d) DHV control; and (e) untreated control. Dotted pooh-" lines prior to 47 hours and between 62 and 70 hours represent periods uui iiio : no observations were made. 18 DECEMBER 1970 DSW 255268 i -s STLCOPCB4060959 birds, were observed. There did not ap DHV control groups would be less than been paid to the role of manganese in pear to be any relation between treat half that in any interaction group on enzyme function, its specific biochemi ment and the presence or absence of the basis of chance alone. cal action in vivo has remained un edema. Histological and residue analy This study illustrates one of the po- clear (5). A variety of evidence, how sis have not been completed at this tentional effects of sublethal concentra ever, has implicated manganese in mi time. tions of chemical pollutants which are tochondrial function, particularly in The suggestions that PCB may render often alluded to, but rarely documented. oxidative phosphorylation (5, 6). In a host more susceptible to certain types It also emphasizes the real differences our laboratory, isolated liver mitochon of infectious agents is the significant that exist between "sublethal" and "no dria from manganese-deficient n.` finding in this study. None of the con effect" concentrations of pollutants. To showed abnormal oxidative phosphory centrations of PCB fed resulted in de the best of our knowledge, other stud lation which appeared to be due to re tectable chemical intoxication even ies in vivo of the possible effects of duced oxidative capacity rather than with the physical stresses of weighing, organochlorine pollutants on the sus to lack of coupling (7). We now report handling, confinement, and crowding ceptibility of a vertebrate host to a viral on P:0 ratios (moles of adenosine during iramportation, transportation it agent have not been reported. triphosphate formed relative to gram self. or relocation in a different environ Milton Friend atoms of oxygen consumed) and rate of ment. However, when the stress of an Daniel O. Trainer oxygen uptake in isolated liver mito infectious agent was added, these sub- Department of Veterinary Science, chondria from manganese-deficient and lethal concentrations appeared to in University of Wisconsin, fluence the resulting mortality rates, Madison 53706 . from pallid mice, and on the fine struc ture of liver tissue as studied with tne causing two- to fourfold increases (14 percent among virus controls versus 35 to 65 percent among groups receiving PCB plus DHV) and reduced incuba tion time (Fig. 1 and Table 2). Similar increases in mortality were obtained with other organochlorine compounds in our laboratory (7). In those studies 30-day-old mallard duck lings which had been fed sublethal con centrations of p,p'-DDT or dieldrin exhibited three- to ninefold increases in mortality over that of DHV controls (6 percent among virus controls versus 19 to 59 percent among groups receiv ing dieldrin plus DHV, and 19 to 40 percent among groups receiving p,p'~ DDT plus DHV). It is unlikely that in all these instances mortality in the References and Notes electron microscope. Female mice maintained in a colony 1. R. W. Riscbrough, P. Ricche, D. B. Peakall, originally derived from a four-way S. G. Herman, M. N. Kirven, Nature 220, 1098 (1968). cross of inbred strains C57B1/16J, 2. C< A. Perming, Ind. Eng. Chem. 22, 1180 (1930). 3. S. Jansen, New Set. 32, 612 (1966). C3H/J, ARK/J, and DBA/2J (8) were given a purified diet (9) contain 4* D. C. Holmes, J. H. Simmons, J. O'G. Tatton, Nature 216, 227 (1967); A. V. Holden and K. ing 1 part manganese per million (de Marsde, ibid., p. 1274; J. H. Koeman, M. C. ficient diet) during pregnancy. Their Tea Noever De Brauw, R. H. DeVes, ibid. 221, 1126 (1969); S. Jensen, A. G. Johnels, M. progeny were maintained on the same Olsson, G. Otterlind, ibid. 224, 247 (1969). diets and were killed as adults for use 5. G. Widmark, J. Ass. Offtc. Agr. Chem. 50, 1069 (1967); L. M. Reynolds, Bull. Environ. in these studies. Hybrid mice of the Contam. Toxicol. 4, 128 (1969). 6. P. P. Levine, in Diseases of Poultry, H. E. Beister and L. H. Schwarte, Ed. (Iowa State same strain were fed a similar purified diet except that it contained 45 parts of Univ. Press, Ames, 1965), p. 838. 7. M, Friend and D. O. Trainer, /. Wildlife Dis.% in press. manganese per million (control diet). Pallid mice (patpa) and their non 8. We thank the Max McGraw Wildlife Founda tion for supplying mallards for this study. Sup ' pallid littermates (C57B1/lOJ-pa) were ported in part by the Patuxent Wildlife Re maintained on a stock diet (10). search Center, Bureau of Sport Fisheries and Wildlife. For mitochondrial preparations, four 16 July 1970; revised 16 October 1970 hybrid deficient, four hybrid control, five pallid, and five nonpallid mice were decapitated; their livers were put into cold 0.25M sucrose, cut into small Liver Mitochondria from Manganese-Deficient and Pallid Mice: Function and Ultrastructure pieces, and washed three times. Mito chondria were separated as previously described (11). Oxygen uptake was de termined by the polarographic assay of Abstract. Oxidative phosphorylation was studied in isolated liver mitochondria oxygen after the method of Chance and from manganese-deficient mice and in those from a mutant strain, pallid. In Williams (12), with /?-hydroxybutyrate mitochondria from manganese-deficient mice, ratios of adenosine triphosphate as the substrate in the reaction medium formed to oxygen consumed were normal, but oxygen uptake was reduced. Elec (Table 1). The oxygen electrode tron microscopy of these mitochondria revealed ultrastructural abnormalities was according to Packer (13). We including elongation and reorientation of cristae. No biochemical or structural calculated P:0 ratios by determining abnormalities were found in mitochondria from pallid mice. the reduction in the amount of added adenosine diphosphate per unit of oxy In a number of animal species a causes congenital ataxia resulting from gen consumed. Adenosine diphosphate striking effect of dietary deficiency of impaired development of otoliths (3). was measured by absorbance at 260 manganese during pregnancy is an ir When the diet of pregnant mutant mice nm (14). The concentration of protein reversible congenital ataxia in the off is supplemented with high amounts of in the mitochondrial suspension, was de spring. The ataxia, characterized by im manganese the otoliths and postural be termined by means of the biuret reac balance and loss of body righting re havior of the offspring are normal. tion (15). flexes, results from abnormal develop Thus, there is a relationship between Median lobular hepatic tissue was ment of the inner ear, with defective the gene pallid and manganese metabo excised from ten manganese-deficient morphogenesis of the otoliths (I, 2). A lism (2, 4). hybrid, five control hybrid, and five mutant gene in mice, pallid (pa), also Although considerable attention has pallid adult female mice for examina- 1316 SCIENCE, VOL. 170 DSW 255269 STLCOPCB4060960